Crystal violet staining solution

PLC-PRF-5 and HepG2 cells subjected to different treatments were added into the top-chamber inserts coated with Matrigel (BD Biosciences). The bottom chamber was filled with 500 µL of medium containing 10% FBS. The cells were cultured at 37 °C for 24 h. The cells transferred through the filter membrane at the bottom of the chambers were washed three times with 1× PBS, fixed in 4% paraformaldehyde (precooled at 4 °C), and stained with crystal violet staining solution (KeyGEN BioTECH). The invading cells were counted under a microscope (Nikon, Japan).

The cells subjected to different treatments were added in top-chamber inserts coated with Matrigel (BD Biosciences, Franklin Lakes, USA). The lower chamber was incorporated with 500 μL of the medium supplemented with 10% FBS and served as a chemotactic agent. After being cultured at 37°C for 24 h, the cells invading the lower chamber were fixed in 4% paraformaldehyde (pre-cooled at 4°C), stained with crystal violet staining solution (KeyGEN, Nanjing, China), and counted under an upright microscope (five fields per chamber).

For material synthesis, 1‐octadecylamine, oleic acid, ethanol, Ca(NO₃)₂, Na₃PO₄, (3‐aminopropyl) triethoxysilane (APTES), ammonia, hydrochloric acid (HCl), sodium hydroxide (NaOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. Dox. N‐Hydroxysulfosuccinimide sodium salt (Sulfo‐NHS) was purchased from Shanghai Macklin Biochemical Co., Ltd. Phosphate buffered saline (PBS), EDC, and Dox were purchased from Sigma‐Aldrich Chemical Reagent Co., Ltd. Anti‐CD44 antibody was purchased from BioLegend (America)
For cell experiments, minimum Essential Medium (MEM), α−minimum essential medium (α−MEM), fetal bovine serum (FBS), and Penicillin–Streptomycin solution (P/S) were purchased from Gibco (America). Cell Plasma Membrane Staining Kit with DiI (Red Fluorescence), Hoechst33342, propidium iodide (PI), and calcein acetoxymethyl ester (Calcein‐AM) were purchased from Beyotime Biotechnology Co., Ltd (China). DAPI was purchased from Abcam. CCK‐8 was purchased from Dojindo (Japan). Crystal violet staining solution and Annexin V‐kFluor488/PI staining assay were purchased from KeyGEN BioTECH (China).

For wound-healing assay, the cells with or without overexpressed Pinin were seeded and cultured until a 90% confluent monolayer was formed. The cells were then scratched by a sterile pipette tip and treated in the FBS-free medium as instructed. Cell migration distances from the scratched area were measured in three randomly chosen fields under a microscope. Wound images were photographed at 0 and 48 h by using a light microscope (Nikon, Japan). For invasion assays, the cells with or without Pinin overexpression were added in top-chamber inserts coated with Matrigel (BD Biosciences, USA). The lower chamber was added with 500 μL of the medium supplemented with 10% FBS and served as a chemotactic agent. After being cultured at 37°C for 24 h, the cells invading into the lower chamber were fixed in 4% paraformaldehyde (precooled at 4°C) and stained with crystal violet staining solution (KeyGEN, Nanjing, China).